Adenovirus expressing p27kip1 suppresses growth of established esophageal carcinoma xenografts

AIM: To investigate the growth suppression of ade novirus expressing p27kip1 on established esophageal tumors in nude mice. METHODS: Esophageal carcinoma xenografts in nude mice were established by tumor tissue mass transplantation. The successfully constructed reco mbinant adenoviral vectors carrying p27kip1 gene (Ad p27kip1) were directly injected into the esophageal tumors in nude mice. Compared to control group, the growth curve of tumor was drawn and the growth inhibition rate of tumor was calculated. The histology of tumors was examined by hematoxylin and eosin (H&E) staining. The expression of p27kip1 and survivin was detected in tumors by immunohistochemical technique. RESULTS: The growth of tumors in gene therapy group with Ad-p27kip1 was obviously suppressed compared to control group (0.42±0.08 g vs 1.17±0.30 g, t=6.39, P<0.01), the inhibition rate of tumor growth reached 64.1%. Pathological detection showed that the tumors in nude mice were poorly differentiated esophageal squamous carcinoma. In addition, the expression of P27kip1 was increased, while the expression of survivin was decreased in tumors after being transfected with Ad-p27kip1. CONCLUSION: p27kip1 gene therapy mediated by adenovirus vector has a significant inhibitory effect on esophageal carcinoma in vivo. Up-regulated p27kip1 expression and down-regulated survivin expression may be its important mechanisms.     

p27kip1腺病毒重组体对人食管癌裸鼠移植瘤生长的抑制作用

D27kip1是新近发现的一种抑癌基因，研究表明p27kip1基因转移能显著抑制食管癌和胃癌细胞生长，该基因疗法在体内实验中是否同样有效值得进一步研究。目的：研究p27kip1腺病毒重组体对人食管癌裸鼠移植瘤生长的抑制作用。方法：将携带人p27kip1基因的重组腺病毒载体导人人食管癌裸鼠移植瘤中．测定肿瘤生长抑制率．免疫组化方法检测移植瘤中p27kip1和生存素(survivin)的表达。结果：经p27kip1基因治疗的裸鼠，肿瘤生长抑制率达64．1％，移植瘤中p27kip1呈高表达．生存素呈低表达。结论：p27kip1腺病毒重组体能显著抑制人食管癌裸鼠移植瘤的生长．上调p27kin1和下调生存素的表达可能是其雷要作用机制。     

p27kip1基因转移对食管癌细胞生存素表达和端粒酶活性的影响

背景：p27kip1是新近发现的一种抑癌基因，早期研究表明p27kip1基因转移能显著抑制食管癌细胞和人食管癌裸鼠移植瘤的生长，表明该基因疗法可能是食管癌治疗的新途径，但其抑癌机制尚未完全阐明。目的：研究p27kip1基因转移对食管癌细胞生存素（survivin）表达和端粒酶活性的影响。从而阐明p27kip1的抑癌机制，为p27kip1基因治疗食管癌提供理论依据。方法：将携带p27kip1基因的重组腺病毒（Ad—p27kip1）和LacZ重组腺病毒（Ad—LacZ）分别转染食管癌细胞系Eca9706，观察细胞形态变化。以免疫细胞化学染色和蛋白质印迹法检测p27kip1和生存素的表达．以端粒重复序列扩增程序（TRAP）聚合酶链反应（PCR）-酶联免疫吸附测定（ELISA）检测端粒酶活性。结果：经Ad—p27kip1转染后，Eta9706细胞变圆，呈葡萄串样聚集以致脱落。细胞p27kip1表达明显增强，生存素表达降低，端粒酶活性显著受抑制。结论：p27kip1基因抑制食管癌细胞生长的作用机制可能与下调生存素表达和抑制端粒酶活性有关。     

Inhibition of adenovirus-mediated p27kip1 gene on growth of esophageal carcinoma cell strain

AIM: To investigate the inhibition of p27kip1 gene on the growth of esophageal carcinoma cell strain (EC9706). METHODS: Recombinant adenovirus Ad-p27kip1 was constructed and transfected into esophageal carcinoma cell EC-9706, and its effect on p27kip1 expression, the growth of esophageal carcinoma cell, DNA replication, protein synthesis, cell multiplication and apoptosis were explored by means of cell growth count, 3H-TdR, 3H-Leucine incorporation, flow cytometry, DNA fragment analysis and TUNEL. RESULTS: Recombinant adenovirus Ad-p27kip1 was successfully constructed with a virus titer of 1.24 X 10(12) pfu/ml. p27kip protein expression increased markedly after EC-9706 transfection, while incorporation quantity of 3H-TdR and 3H-Leucine decreased significantly. The growth of esophageal carcinoma cell was inhibited obviously. Testing of flow cytometry displayed a typical apoptosis peak, and DNA gel electrophoresis showed a typical apoptosis ladder. TUNEL showed the apoptosis rate of Ad-p27kip1 group and control group to be 37.3% and 1.26% (P<0.001) respectively. CONCLUSION: Ad-p27kip1 can inhibit the growth and multiplication of esophageal carcinoma cells and induce apoptosis. Therefore, enhanced p27kip1 expression may be a new way to treat esophageal carcinoma.     

突变型p27对大肠癌裸鼠模型肿瘤生长及转移的作用

目的研究外源性突变型p27(p27mt)基因对大肠癌体内抑制作用。方法以腺病毒为载体,采用细菌内同源重组方法制备Ad-p27mt,应用细胞移植法把人大肠癌SW480细胞接种于BALB/c裸鼠皮下,构建荷大肠癌裸鼠模型,将已成功构建的Ad-p27mt及Lac-Z采用瘤体内直接注射法注射到瘤体内,绘制肿瘤生长曲线,计算肿瘤生长抑制率,进行细胞凋亡及MMP-9检测。结果Ad-p27mt基因治疗组肿瘤生长明显受到抑制,有更强促凋亡作用,MMP-9表达明显降低,与Lac-Z、对照组比较,有显著性差异。结论Ad-p27mt基因对大肠癌肿瘤生长有明显抑制作用并能减少其转移。     

Impact of p27mt gene on transplantation model of human colorectal cancer in nude mice

AIM： To investigate the inhibitory and anti-metastatic effect of mutant p27 gene （p27mt） on the growth of colorectal cancer xenografts in nude mice and its underlying mechanism. METHODS： Inhibitory effect of p27mt gene on the growth of colorectal cancer xenografts was determined by measurement of tumor size before and after direct intra-tumoral injection of Ad-p27mt in a pre-established transplantation model of human colorectal cancer in nude mice. Cell cycle and apoptosis were detected by flow cytometry performed on single-cell suspension from an isolated tumor. Expression of MMP-9 in tumor tissue was detected by immunohistochemistry. RESULTS： The average sizes of transplantation tumors were 1.94 ± 0.67 cm^3, 2.75 ± 0.83 cm^3 and 3.01 ± 0.76 cm^3 in the Ad-p27mt, Ad-LacZ and control groups, respectively （P 〈 0.05）. The average proliferation rates were 37.34% ± 1.45%, 53.16% ± 3.27% and 54.48% ± 2.43%, in the Ad-p27mt, Ad-LacZ and control groups, respectively （P 〈 0.05）. The average apoptosis rates were 19.79% ± 3.32%, 6.38% ± 4.91% and 7.25% ± 5.20% in the Ad-p27mt, Ad-LacZ and control groups, respectively （P 〈 0.01）. The average MMP-9 expression rates were 20%, 75% and 66.7% in the Ad-p27mt, Ad-LacZ and control groups, respectively （P 〈 0.01）. CONCLUSION： p27mt inhibits the growth of transplanted tumor by blocking the proliferation of cancer xenografts and by promoting apoptosis of transplantated tumor cells, as well as decrease transplanted tumor metastasis.     

Molecular therapy with recombinant antisense c-myc adenovirus for human gastric carcinoma cells in vitro and in vivo

BACKGROUND AND AIMS: This study used a recombinant antisense c-myc adenovirus (Ad-ASc-myc) to evaluate how alterations of c-myc expression in the SGC7901 human gastric carcinoma cells could influence the proliferation, apoptosis and the growth of human gastric tumors in nude mice. METHODS: The human gastric carcinoma cell line, SGC7901, treated with Ad-ASc-myc or adenovirus recombinants carrying LacZ gene (Ad-LacZ) were analyzed by using X-gal stain, MTT, DNA ladder, TUNEL assay, flow cytometric analysis, polymerase chain reaction and western blot in vitro. The tumorigenicity and experimental therapy in nude mice models were assessed in vivo. RESULTS: The Ad-ASc-myc could strongly inhibit cell growth and induce apoptosis in SGC7901 cells. The proliferation of the Ad-ASc-myc-infected SGC7901 cells was reduced by 44.1%. The mechanism of killing gastric carcinoma cells by Ad-ASc-myc was found to be apoptosis, which was detected by the use of a DNA ladder, TUNEL and flow cytometric analysis. Infection of Ad-ASc-myc in nude mice showed that all three mice failed to form tumors from the 7 to 30 day period, compared with injection of Ad-LacZ and parent SGC7901 cells. Experimental therapy on the nude mice bearing subcutaneous tumors of SGC7901 cells showed that intratumor instillation of Ad-ASc-myc inhibited the growth of the tumors. Recombinant antisense c-myc adenovirus-treated tumors were inhibited by 68.9%, compared with tumors injected with Ad-LacZ and control (LacZ and phosphate-buffered saline). CONCLUSION: The expression of Ad-ASc-myc can inhibit growth and induce apoptosis of gastric cancer cells in vitro and in vivo and thus is a potential clinical utility in gene therapy for the treatment of gastric carcinoma.     

p16-INK4alpha重表达对人食管癌细胞系EC109生长的抑制作用

目的探讨用腺病毒介导野生型的p16基因在食管癌细胞系表达,研究重表达野生型p16基因对食管癌细胞系EC109生长的抑制作用,为寻找食管癌致癌机制的研究及其基因治疗提供理论基础.方法用基因重组技术,将pcDNA3-p16中的野生型p16基因用Kpn I/BamH I进行双酶切,克隆入腺病毒表达载体pAdCMV中,将重组质粒pad-CMV-p16与腺病毒质粒JM17用Lipofectamime 2000共转染293细胞,产生重组腺病毒.用重组腺病毒感染人食管癌细胞系EC109,在感染后的不同时段,用免疫荧光、打点杂交方法检测p16基因在细胞中的表达用MTT法及流式细胞仪观察p16基因的表达对食管癌细胞株生长的影响.结果经酶切鉴定野生型p16基因克隆入腺病毒表达载体中,与JM17共转染293细胞后,可产生具有感染活性的重组腺病毒,用重组腺病毒感染食管癌细胞株EC109细胞后,可抑制食管癌细胞的生长,同对照组相比其最大抑制率可达52.7%.Multipcycle分析软件分析对细胞周期影响表明,处于G0～G1期的细胞为41%～63%感染后的细胞经Dot blot和Westernblot杂交证实,有外源的p16mRNA及蛋白的表达.结论重组腺病毒可将野生型p16基因导入人食管癌细胞系EC109中,野生型p16基因在p16基因功能缺失的细胞中重表达能抑制癌细胞恶性生长.p16基因功能缺失是食管癌致癌因素之一     

重组腺病毒介导的人p27kip1基因高表达对体外培养的大鼠主动脉血管平滑肌细胞迁移的影响

目的　探讨重组腺病毒介导的p2 7kip1基因及其蛋白产物高表达对血管平滑肌细胞 (VSMCs)迁移的抑制作用。方法　将含人p2 7kip1cDNA的重组腺病毒 (Adhp2 7kip1)及含 β 半乳糖苷酶基因的重组腺病毒 (AdLacZ)在体外转染原代大鼠主动脉VSMCs,用Westernblot及Boyden趋化小室检测外源性p2 7kip1蛋白在细胞内的表达及对VSM Cs迁移的影响。结果　转染后 2 4h ,Adhp2 7kip1转染的VSMCs内p2 7kip1蛋白高表达 ,而AdLacZ转染的VSMCs内仅显示极低水平的内源性p2 7kip1蛋白表达 ;Boyden趋化小室检测显示未转染的VSMCs、AdLacZ及Adhp2 7kip1转染的VSMCs血清诱导后的迁移细胞数分别为 139± 2 6、10 6± 16及 6 8± 14。结论　外源性p2 7kip1基因及蛋白产物在VSMCs内高表达可显著抑制VSMCs的迁移     

腺病毒介导的p53抑制肝癌生长的实验研究

目的探讨外源性野生型Ｐ５３（Ｗｉｌｄ－ｔｙｐｅ ｐ５３, ＷＴ－ｐ５３）基因对肝癌细胞生长的影响。方法通过表达ＷＴ－ｐ５３的重组腺病毒载体（Ａｄ－ｐ５３）,将ｐ５３基因导入肝癌细胞株中,用ＭＴＴ法检测Ａｄ－ｐ５３对各细胞株生长特性的影响,流式细胞仪分析各组细胞中ＤＮＡ含量及凋亡的比例。建立裸小鼠移植瘤模型后,瘤内注射Ａｄ－ｐ５３。最后处死动物,移植瘤称重, ｗｅｓｔｅｒｎ　ｂｌｏｔ检测ｐ５３和ｐ２１蛋白的表达情况。结果外源性ｐ５３基因的导入可导致肝癌细胞生长的明显抑制,表现为既诱导细胞凋亡,又诱导细胞周期阻滞。瘤内注射Ａｄ－ｐ５３后移植瘤生长明显受抑制,并出现瘤组织中ｐ５３和ｐ２１蛋白表达的上调。结论腺病毒介导的ｐ５３基因治疗能有效控制肝癌的生长。     

Effect of mutant p27kip1 gene on human cholangiocarcinoma cell line, QBC939

AIM:To investigate the effects of exogenously mutated p27kip1 (p27) on proliferation and apoptosis of human cholangiocarcinoma cell line,QBC939 in vivo.METHODS:Adenviral vectors were used to transfect mutated p27 cDNA into human QBC939 cell line.Expression of p27 was detected by RT-PCR.Western blot.Cell growth,morphological change,cell cycle,apoptosis and cloning formation were determined by MTT assay and flow cytometry.RESULTS:The expression of p27 protein and mRNA was increased significantly in QBC939 cell line transfected with Ad-p27mt.The transfer of Adp27mt could significantly inhibit the growth of QBC939cells,decrease the cloning formation rate and induce apoptosis,p27 over expression caused cell cycle arrest at G0/G1 phase 72 h after infection with Adp27mt.CONCLUSION:p27 may cause cell cycle arrest at G0/G1 phase and subsequently lead to apoptosis.Recombinant adenovirus expressing mutant p27 may be potentially useful in gene therapy for cholangiocarcinoma.     

Antitumor bioactivity of adenovirus-mediated p27mt in colorectal cancer cell line SW480

AIM： To explore the antitumor bioactivity of adenovirusmediated mutant type p27^kip1 gene in a colorectal cancer cell line SW480. METHODS： We constructed recombinant adenovirus vector expressing a mutant type p27^kip1 gene （ad- p27mt）, with mutation of Thr-187/Pro-188 （ACGCCC） to Met-187/Ile-188 （ATGATC）, and transduced into SW480 cells. Then we detected expression of p27, Bcl-2 and Bax protein in the transductants by Western blotting, cell cycle of transductants by a digital flow cytometric system, migrating potential with Boyden Chamber and SW480 tumor cell growth inhibition in vitro and in vivo. RESULTS： We found that a recombinant adenovirus vector of expressing ad-p27mt, with mutation of Thr-187/Pro-188 （ACGCCC） to Met-187/Ile-188 （ATGATC） has potent inhibition of SW480 tumor cell growth in vitro and in vivo. Furthermore, ad-p27mt induced cell apoptosis via regulating bax and bcl-2 expressions, and G1/S arrest in SW480 cells and inhibited cell migration. CONCLUSION： ad-p27mt has a strong anti-tumor bioactivity and has the potential to develop into new therapeutic agents for colorectal cancer.     

携带人Endostatin基因的腺病毒治疗胰腺癌移植瘤

目的 观察携带人Endostatin基因的重组腺病毒载体Ad-hEnd对裸鼠胰腺癌移植瘤的治疗作用.方法 将胰腺癌细胞株SW1990细胞皮下注射建立裸鼠移植瘤模型,随机分为Ad-hEnd组、报告基因LacZ重组腺病毒组(Ad-LacZ组)和对照组,每组8只.重组腺病毒200 μl瘤内注射,隔日1次,共4次.观察移植瘤的生长情况,免疫组化染色检测血管内皮生长因子(VEGF)的表达和微血管密度(MVD),原位缺口末端标记法(TUNEL)检测肿瘤细胞凋亡.结果 移植瘤成瘤率100%.治疗后4周,Ad-hEnd组、Ad-LacZ组和对照组移植瘤体积分别为(921.9±279.7)mm3、(2804.4±553.5)mm3和(3040.6±487.6)mm3;瘤重分别为(1.19±0.18)g、(2.38±0.42)g和(2.41±0.47)g;VEGF表达阳性率分别为(36.3±7.1)%、(81.2±6.6)%和(79.4±6.2)%;MVD分别为12±4、27±5和25±6;细胞凋亡率分别为(31.2±5.4)%、(9.4±4.9)%和(8.5±3.7)%.与Ad-LacZ组和对照组比较,Ad-hEnd组以上各项指标均有显著性差异(P＜0.01);Ad-LacZ组和对照组之间的差异无统计学意义.结论 重组腺病毒介导的hEndostatin基因可抑制胰腺癌的生长和血管生成,促进肿瘤细胞凋亡,可用于胰腺癌抗血管生成的基因治疗.     

Adenovirus expressing mutant p27kip1 enhanced apoptosis against cholangiocarcinoma than adenovirus-p27kip1 wild type

BACKGROUND/AIMS: The prognosis of cholangiocarcinoma is extremely poor despite the aggressive multidisciplinary cancer therapies that have been used clinically. Thus, it is imperative to develop new and effective treatments, such as gene therapy in order to treat this disease. p53 is the most common target for cancer gene therapy treatment. However, cholangiocarcinoma has a low frequency of p53 mutation, which makes this protein a poor candidate for gene therapy in this disease and another suitable gene therapy target must be found. p27kip1 is a universal cyclin-dependent kinase (CDK) inhibitor that blocks cell cycle progression and inhibits proliferation. Our previous reports have demonstrated the role of p27kip1 in the induction of apoptosis using a recombinant adenoviral vector expressing p27kip1 (Adp27) in several different human cancer cells. p27kip1 is regulated by two mechanisms composed of a ubiquitin-proteasome system and proteolytic processing system that requires the phosphorylation of p27kip1 on Thr-187 by the cyclinE/Cdk2 complex followed by proteolytic degradation. METHODOLOGY: In this study, we focused our aim on the degradation of p27kip1 protein mediated by a recombinant adenoviral expressing a mutant p27kip1 (Adp27-mt), which has a mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188 (ATGATC). RESULTS: We observed that the mutated p27kip1 markedly inhibited ubiquitination and the subsequent degradation of p27kip1 when compared to wild type p27kip1. Consequently, we found that mutated p27kip1 induced a stronger induction of apoptosis and cell growth inhibition than wild type p27kip1 in cholangiocarcinoma cell lines TFK-1 and HuCCT-1. Furthermore, we demonstrated that Adp27-mt mainly caused G2/M arrest in the cell cycle progression and a decreased cyclinB1 and Cdc2 protein where as Adp27-wt mediated a G1/S arrest at 48 hours after infection. CONCLUSIONS: In this study, we showed that adenoviral vector expression of mutant p27kip1 protein inhibited degradation by the ubiquitin-proteasome system and strongly induced apoptosis and cell growth inhibition compared to wild type p27kip1. Thus recombinant adenovirus expressing mutant p27kip1 may be potentially useful for gene therapy against cholangiocarcinoma.     

Trans-arterial gene therapy for hepatocellular carcinoma in a rabbit model

AIM： To study the effect of adenovirus （Ad）-p53 gene therapy on hepatocellular carcinoma （HCC） in a rabbit model.
METHODS： VX2 tumor was grown in the liver of 24rabbits. Animals were divided into four groups： group A receiving trans-arterial gene therapy （Ad-p53） only,group B receiving combined Ad-p53 therapy and transarterial embolization （lipiodol）, group C receiving transarterial chemoembolization （lipiodol ＋ mitomycin C）,control group （D） receiving sodium chloride. Tumor volume （V1） was measured by using MRI （d 13）.Interventional procedure was applied （d 14）.Tumor volume （V2） was assessed by MRT （d 21） and the mean ratio （V2/V1） was calculated. After the second MRI,specimens of the liver were abstained and examined immunohistochemically using mutant-type p53 antibody.The positive expression was scored.
RESULTS： Compared with control group （（^-x） = 3.14± 0.64）, therapeutic groups all showed a significant decrease in the tumor growth ratio （P 〈 0.05）. A slight difference was found between group A （（^-x） =2.35 ±0.59） and group B （（^-x） = 1.75 ± 0.28） （P = 0.048）. Nostatistically significant difference was observed between group B and group C （（^-x） = 2.00 ± 0.44）. The positive expression rate of mutant-type p53 was the lowest in group B and significantly different between group A and group C （P 〈 0.05）.Compared to the control subjects, groups A and C both showed a decrease in the expression of mutant-type p53, but there was no significant difference between them.
CONCLUSION： Trans-arterial Ad-p53 gene therapy can reduce tumor growth of HCC in rabbit model.     

Effect of mutant p27(kipl) gene on human cholangiocarcinoma cell line, QBC(939)

AIM：To investigate the effects of exogenously mutated p27^kip1 （p27） on proliferation and apoptosis of human cholangiocarcinoma cell line, QBC939 in vivo.METHODS： Adenviral vectors were used to transfect mutated p27 cDNA into human QBC939 cell line. Expression of p27 was detected by RT-PCR. Western blot. Cell growth, morphological change, cell cycle, apoptosis and cloning formation were determined by MTT assay and flow cytometry.RESULTS： The expression of p27 protein and mRNA was increased signifi cantly in QBC939 cell line transfected with Ad-p27mt. The transfer of Ad-p27mt could signifi cantly inhibit the growth of QBC939 cells, decrease the cloning formation rate and induce apoptosis. p27 over expression caused cell cycle arrest at G0/G1 phase 72 h after infection with Ad-p27mt.CONCLUSION： p27 may cause cell cycle arrest at G0/G1 phase and subsequently lead to apoptosis. Recombinant adenovirus expressing mutant p27 may be potentially useful in gene therapy for cholangiocarcinoma.     

加载野生型p53基因鼠树突细胞的抗肿瘤作用

目的 观察加载了野生型p53基因的树突细胞(DC)对不同位点p53基因突变肿瘤得庖咧瘟谱饔?方法通过锥虫蓝染色、同种异体混合白细胞反应及流式细胞仪检测DC细胞表面分子,评估腺病毒(Ad)-p53感染DC是否影响DC的免疫功能.以Ad或转导了野生型p53基因的Ad分别感染骨髓De(Ad-DC和Ad-p53-DC)后,静脉注射免疫C57BL/6小鼠各5只,分离脾细胞,采用标准6 h51 Cr释放试验测定其诱导不同肿瘤细胞系(MethA、D459和P815)细胞毒性T淋巴细胞(CTL)的杀伤活性;效应细胞(Ad-p53-DC免疫后的小鼠脾细胞)和靶细胞(Ad-p53-P815和D459)孵育时分别加入抗CD4抗体或抗CD8抗体,观察CTL的活性变化.使用MethA和D459肿瘤细胞系得到不同的荷瘤鼠,于肿瘤形成前后分别使用Ad-p53-DC免疫,Ad-DC对照,当实体瘤三维直径之和＞20 cm时处死小鼠,用生存曲线评估Ad-p53-DC免疫的预防或治疗作用.结果 (1)Ad-p53-DC免疫诱导的抗Ad-p53-P815、D459和MethA的CTL反应(效应细胞:靶细胞=50:1)分别为(27.8±3.4)%、(23.5±2.7)%及(58.3±9.2)%,与Ad-DC免疫诱导的反应[(9.3±1.8)%、(4.6±1.0)%及(23.5 ±3.7)%]相比,差异有统计学意义(td值分别为5.79、3.68、5.02,均P＜0.05).Ad-p53-DC免疫小鼠T淋巴细胞与靶细胞Ad-p53-P815或D459的CTL活性,抗CD4组[(59.8 ±4.6)%、(18.9±2.4)%]与无抗体组[(64.4±6.3)%、(22.2±3.0)%]相比,差异无统计学意义(td值分别为0.84、0.91,均P＞0.05),而抗CD8组[(26.7±2.8)%、(6.1±1.2)%]差异有统计学意义(td值分别为9.03、7.67,均P＜0.05).抗CD8组与抗CD4组比较,差异有统计学意义(td值分别为8.79、9.18,均P＜0.05).(2)Ad-p53-DC和Ad-DC静脉注射2次免疫小鼠后,分别以D459细胞或MethA肉瘤细胞荷瘤20只小鼠.在Ad-p53-DC免疫组分别有14只和16只小鼠肿瘤的生长得到完全抑制,与Ad-DC免疫组比较差异有统计学意义(x2值分别为6.72、5.86,P＜0.05).皮下接种小鼠D459后,Ad-p53-DC免疫治疗组的小鼠肿瘤生长速度比Ad-DC组延缓2周左右,二组比较差异有统计学意义(x2 值为9.48,P＜0.05).结论 Ad-p53-DC可诱导抗MethA、P815和D459靶细胞的由CD8+T淋巴细胞介导的CTL反应,并抑制鼠体内肿瘤细胞的形成和生长.     

转染kiss-1基因对人食管癌EC9706细胞裸鼠移植瘤的抑制作用

目的:研究转染kiss-1基因对人食管癌EC9706细胞裸鼠皮下移植瘤的作用,探讨其在食管癌基因治疗中的可行性和特异性.方法:在食管癌细胞系EC9706中转染kiss-1基因,经G418筛选,建立稳定高表达Kiss-1蛋白的细胞系.稳定表达该基因的细胞为转染kiss-1基因组,转染空质粒细胞及未处理细胞为对照组,建立裸鼠荷瘤模型;监测肿瘤生长变化,HE染色观察肿瘤病理学变化,RT-PCR、Western blot方法检测kiaa-1 mRNA和蛋白变化.结果:转染kiss-1基因组肿瘤生长受到显著抑制:HE染色显示转染kiss-1基因组及转染空质粒纽肿瘤组织内坏死均较空白对照组多;RT-PCR、Western blot结果表明转染kiss-1基因组裸鼠肿瘤组织kiss-1 mRNA和蛋白表达均显著升高,三组间比较差异具有统计学意义(F=72.685,24.807,均P<0.05).结论:转染kiss-1基因能抑制人食管癌EC9706细胞裸鼠皮下移植瘤的形成,且能有效上调kiss-1 mRNA和蛋白的表达,可为食管癌的基因治疗提供新的靶点、开辟新的思路.     

鸟苷酸环化酶C基因沉默对人胃癌裸鼠皮下种植瘤的影响

目的:探讨鸟苷酸环化酶C(GC-C)基因沉默对人胃癌裸鼠皮下种植瘤的影响及其机制.方法:将SGC-7901细胞(SGC-7901组)、空质粒转染的细胞(PRNA组)、GC-C基因稳定沉默的细胞(GC-C-shRNA组)悬液先在裸鼠皮下接种成瘤,再取瘤组织块分别接种到裸鼠皮下,建立3种胃癌裸鼠皮下种植瘤模型.观察裸鼠一般情况、肿瘤的成瘤率、生长速度,描绘肿瘤的生长曲线,计算肿瘤的抑瘤率;采用实时荧光定量PCR(RFQ-PCR),Western blot和免疫组织化学法检测GC-C和趋化因子受体4(CXCR4)在各组移植瘤组织中的表达.结果:与SGC-7901组和PRNA组比较,GC-C-shRNA组裸鼠移植瘤生长速度明显减慢、体积明显变小(抑制率分别为33.7%、33.2%),肿瘤异型性、坏死程度明显降低,差异具有统计学意义(均P<0.05),且移植瘤组织中GC-C和CXCR4 mRNA和蛋白均明显下调(GC-CmRNA:7.47±1.70 vs 11.18±0.60,11.28±0.85;GC-C蛋白:0.52±0.15 vs 1.04±0.19,1.03±0.24;CXCR4蛋白:0.67±0.13 vs 1.02±0.21,1.03±0.23,均P<0.05).结论:GC-C基因稳定沉默在体内能保持稳定,且能抑制裸鼠移植瘤生长,该过程可能与CXCR4下调有关.